Ozone-induced airway epithelial cell death, the neurokinin-1 receptor pathway, and the postnatal developing lung.

Children are uniquely susceptible to ozone because airway and lung growth continue for an extensive period after birth. Early-life exposure of the rhesus monkey to repeated ozone cycles results in region-specific disrupted airway/lung growth, but the mediators and mechanisms are poorly understood. Substance P (SP), neurokinin-1 receptor (NK-1R); and nuclear receptor Nur77 (NR4A1) are signaling pathway components involved in ozone-induced cell death. We hypothesize that acute ozone (AO) exposure during postnatal airway development disrupts SP/NK-1R/Nur77 pathway expression and that these changes correlate with increased ozone-induced cell death. Our objectives were to 1) spatially define the normal development of the SP/NK-1R/Nur77 pathway in conducting airways; 2) compare how postnatal age modulates responses to AO exposure; and 3) determine how concomitant, episodic ozone exposure modifies age-specific acute responses. Male infant rhesus monkeys were assigned at age 1 mo to two age groups, 2 or 6 mo, and then to one of three exposure subgroups: filtered air (FA), FA+AO (AO: 8 h/day × 2 days), or episodic biweekly ozone exposure cycles (EAO: 8 h/day × 5 days/14-day cycle+AO). O3 = 0.5 ppm. We found that 1) ozone increases SP/NK-1R/Nur77 pathway expression in conducting airways, 2) an ozone exposure cycle (5 days/cycle) delivered early at age 2 mo resulted in an airway that was hypersensitive to AO exposure at the end of 2 mo, and 3) continued episodic exposure (11 cycles) resulted in an airway that was hyposensitive to AO exposure at 6 mo. These observations collectively associate with greater overall inflammation and epithelial cell death, particularly in early postnatal (2 mo), distal airways.

Postnatal exposure history and airways: oxidant stress responses in airway explants.

Postnatally, the lung continues to grow and differentiate while interacting with the environment. Exposure to ozone (O(3)) and allergens during postnatal lung development alters structural elements of conducting airways, including innervation and neurokinin abundance. These changes have been linked with development of asthma in a rhesus monkey model. We hypothesized that O(3) exposure resets the ability of the airways to respond to oxidant stress and that this is mediated by changes in the neurokinin-1 receptor (NK-1R). Infant rhesus monkeys received episodic exposure to O(3) biweekly with or without house dust mite antigen (HDMA) from 6 to 12 months of age. Age-matched monkeys were exposed to filtered air (FA). Microdissected airway explants from midlevel airways (intrapulmonary generations 5-8) for four to six animals in each of four groups (FA, O(3), HDMA, and HDMA+O(3)) were tested for NK-1R gene responses to acute oxidant stress using exposure to hydrogen peroxide (1.2 mM), a lipid ozonide (10 µM), or sham treatment for 4 hours in vitro. Airway responses were measured using real-time quantitative RT-PCR of NK-1R and IL-8 gene expression. Basal NK-1R gene expression levels were not different between the exposure groups. Treatment with ozonide or hydrogen peroxide did not change NK-1R gene expression in animals exposed to FA, HDMA, or HDMA+O(3). However, treatment in vitro with lipid ozonide significantly increased NK-1R gene expression in explants from O(3)-exposed animals. We conclude that a history of prior O(3) exposure resets the steady state of the airways to increase the NK-1R response to subsequent acute oxidant stresses.

Postnatal lung development of rhesus monkey airways: cellular expression of Clara cell secretory protein.

Clara cell secretory protein (CCSP) is a protective lung protein that is believed to have antioxidant, immunomodulatory, and anticarcinogenic properties. Evidence suggests that CCSP is involved in mitigating many lung disease states during development including asthma. This study's rationale is to define the distribution and abundance of CCSP in the airway epithelium of the rhesus monkey during postnatal lung development using carefully controlled site-specific morphometric approaches in defined airway regions. Immunoreactive CCSP was found in nonciliated cells and mucous cells, including glands, throughout the airway epithelium at all ages, with proximal and mid-level airways having the highest labeling. Overall airway CCSP levels were low at 1 week and 1 month, doubled between 1 and 3 months, and changed little from 3 months to 3 years. Thus, the critical developmental window for CCSP expression to reach adult levels in the rhesus conducting airways occurs between 1 and 3 months of age.

ß-Adrenergic Inhibition Prevents Action Potential and Calcium Handling Changes during Regional Myocardial Ischemia.

ß-adrenergic receptor (ß-AR) blockers may be administered during acute myocardial infarction (MI), as they reduce energy demand through negative chronotropic and inotropic effects and prevent ischemia-induced arrhythmogenesis. However, the direct effects of ß-AR blockers on ventricular electrophysiology and intracellular Ca2+ handling during ischemia remain unknown. Using optical mapping of transmembrane potential (with RH237) and sarcoplasmic reticulum (SR) Ca2+ (with the low-affinity indicator Fluo-5N AM), the effects of 15 min of regional ischemia were assessed in isolated rabbit hearts (n = 19). The impact of ß-AR inhibition on isolated hearts was assessed by pre-treatment with 100 nM propranolol (Prop) prior to ischemia (n = 7). To control for chronotropy and inotropy, hearts were continuously paced at 3.3 Hz and contraction was inhibited with 20 µM blebbistatin. Untreated ischemic hearts displayed prototypical shortening of action potential duration (APD80) in the ischemic zone (IZ) compared to the non-ischemic zone (NI) at 10 and 15 min ischemia, whereas APD shortening was prevented with Prop. Untreated ischemic hearts also displayed significant changes in SR Ca2+ handling in the IZ, including prolongation of SR Ca2+ reuptake and SR Ca2+ alternans, which were prevented with Prop pre-treatment. At 5 min ischemia, Prop pre-treated hearts also showed larger SR Ca2+ release amplitude in the IZ compared to untreated hearts. These results suggest that even when controlling for chronotropic and inotropic effects, ß-AR inhibition has a favorable effect during acute regional ischemia via direct effects on APD and Ca2+ handling.

Fetal exposure of rhesus macaques to bisphenol a alters cellular development of the conducting airway by changing epithelial secretory product expression.

BACKGROUND: Bisphenol A (BPA) exposure early in life results in organizational changes in reproductive organs, but the effect of BPA on conducting airway cellular maturation has not been studied. Late gestation is characterized by active differentiation of secretory cells in the lung epithelium. OBJECTIVE: We evaluated the hypothesis that BPA exposure disrupts epithelial secretory cell development in the fetal conducting airway of the rhesus macaque. METHODS: We exposed animals to BPA during either the second (early term) or the third (late term) trimester. There were four treatment groups: a) sham control early term, b) sham control late term, c) BPA early term (BPA-early), and d) BPA late term (BPA-late). Because cellular maturation occurs nonuniformly in the lung, we defined mRNA and protein expression by airway level using microdissection. RESULTS: BPA exposure of the dam during late term significantly accelerated secretory cell maturation in the proximal airways of the fetus; both Clara cell secretory protein (CCSP) and MUC5AC/5B mRNA and protein expression increased. CONCLUSIONS: BPA exposure during late gestation accelerates secretory cell maturation in the proximal conducting airways. We identified a critical window of fetal susceptibility for BPA effects on lung epithelial cell maturation in the third trimester. This is of environmental health importance because increases in airway mucins are hallmarks of a number of childhood lung diseases that may be affected by BPA exposure.

Ozone exposure alters serotonin and serotonin receptor expression in the developing lung.

Ozone, a pervasive environmental pollutant, adversely affects functional lung growth in children. Animal studies demonstrate that altered lung development is associated with modified signaling within the airway epithelial mesenchymal trophic unit, including mediators that can change nerve growth. We hypothesized that ozone exposure alters the normal pattern of serotonin, its transporter (5-HTT), and two key receptors (5-HT2A and 5-HT4), a pathway involved in postnatal airway neural, epithelial, and immune processes. We exposed monkeys to acute or episodic ozone during the first 2 or 6 months of life. There were three exposure groups/age: (1) filtered air, (2) acute ozone challenge, and (3) episodic ozone + acute ozone challenge. Lungs were prepared for compartment-specific qRT-PCR, immunohistochemistry, and stereology. Airway epithelial serotonin immunopositive staining increased in all exposure groups with the most prominent in 2-month midlevel and 6-month distal airways. Gene expression of 5-HTT, 5-HT2AR, and 5-HT4R increased in an age-dependent manner. Overall expression was greater in distal compared with midlevel airways. Ozone exposure disrupted both 5-HT2AR and 5-HT4R protein expression in airways and enhanced immunopositive staining for 5-HT2AR (2 months) and 5-HT4R (6 months) on smooth muscle. Ozone exposure increases serotonin in airway epithelium regardless of airway level, age, and exposure history and changes the spatial pattern of serotonin receptor protein (5-HT2A and 5-HT4) and 5-HTT gene expression depending on compartment, age, and exposure history. Understanding how serotonin modulates components of reversible airway obstruction exacerbated by ozone exposure sets the foundation for developing clinically relevant therapies for airway disease.